DESCRIPTION (from the application): We recently demonstrated that neuronal ABeta formation is reduced by estrogen. Additionally it was shown that insulin stimulates ABeta secretion, which causes reduced levels of intracellular ABeta. An understanding of the molecular mechanism by which these hormones regulate BetaAPP processing and ABeta formation could be important in elucidating the causes of AD and for developing effective therapeutics. Estrogen has been shown to stimulate the secretion of various proteins, such as prolactin from neuroendocrine cells. Insulin signal transduction has been shown to increase movement of glucose transporter-containing vesicles from post-TGN compartments to the plasma membrane. Insulin has also been shown to stimulate trafficking of leptin from the endoplasmic reticulum (ER) to the Golgi. It is therefore possible that gonadal hormones and insulin affect APP processing by mediating BetaAPP trafficking. The ABeta generating enzyme, Beta-secretase (BACE), was recently cloned; the other enzyme required for ABeta generating activity, Beta-secretase, has not been unequivocally identified, although presenilin has been suggested to closely modulate its activity, and may yet be shown to contain the catalytic site of Beta-secretase Therefore, understanding the effects of hormones on the secretase activities will be important for developing therapies aimed at ABeta reduction. In Specific Aim I we propose to examine the effect of gonadal steroids and insulin on the formation of various species of ABeta and BetaAPP derivatives in specific subcellular compartments, and on regulation of BetaACE expression and activity in neuronal cultures. We also propose to explore hormonal effects on the proteolytic cleavage of presenilins and the interaction of presenilins with BetaAPP and its metabolites. In Specific Aim II we propose to examine the effect of these hormones on intracellular trafficking of BetaPP/ABeta within the secretor) and endocytic compartments using cell-free reconstituted systems derived from neuronal cells. In Specific Aim III we propose to identify and characterize potential cytosolic proteins and membrane factors that modulate BetaAPP trafficking and ABeta formation. In Specific Aim IV we propose to investigate the role of steroid hormones and insulin in regulating intraorganellar calcium and pH using a cell-free system in which pH and calcium concentration can be readily manipulated.